Why does deuterium replace hydrogen

Imagine you are a racer, but your slender cousin always starts 10 meters ahead of you, partway up the hill. She takes off for the summit, and she always surmounts the peak before you do and then rapidly begins her descent. Your race time will always be slower than hers. Reacting organic molecules can face similar challenges. They must surmount high barriers that slow their passage to products. Many of these reactions involve breaking carbonhydrogen bonds.

In such cases, mass makes a difference. During chemical reactions, molecules must overcome energy barriers, which can be thought of as hill-like obstacles, as they transform from reactants to products. But if a reaction involves breaking a bond between carbon and hydrogen, the lightest hydrogen isotope H always has a head start over heavier hydrogen isotopes, such as deuterium D. The kinetic isotope effect is a tool that chemists can exploit to understand how reactions work, to alter the course of reactions, and to change how drugs react within the human body.

Chemists can use kinetic isotope effects to hack into chemical reactions. If replacing hydrogen with deuterium slows a reaction, then they know that bond between carbon and either hydrogen or deuterium is likely broken in or before the rate-determining transition state.

The transition state sits at the summit of the tallest hill and lasts for mere femtoseconds. Theoretical chemists like me and researchers in my group can help by providing new hypotheses that we generate from computational models. We can calculate kinetic isotope effects from the properties of a transition state and then compare these predicted results to kinetic isotope effects measured from reactions in flasks. If the measured and predicted kinetic isotope effects do not match, our hypothesis is faulty.

If the measured and predicted results match, then the computed transition state structure is considered reasonable. Organic chemists have most often used kinetic isotope experiments to decide between competing proposals for the mechanisms, or paths, of reactions. With more precise knowledge of when and why bonds break and form, chemists can design new strategies for assembling everything from drugs to plastics. When they used a farnesyl diphosphate that included deuterium rather than hydrogen, they observed a dramatically different ratio of the two products.

That meant that the product-determining step in the reaction must involve breaking a carbon-hydrogen bond: The green pathway was wrong, but the blue pathway was reasonable. Our group wanted to test the validity of our theoretical and computational methods for interrogating reaction mechanisms, and we decided to look at the formation of pentalenene from farnesyl diphopshate in the presence of an enzyme called pentalenene synthase see figure above.

The structure was intriguingly complex, and experimental chemists had studied the system, giving us data against which we could compare our theoretical models.

The deuterium exchange can be seen as the progressive mass spectra are compared with the immediately preceding spectra. The exchange can be monitored through the centroided mass of a group of peaks, or by dissecting each peak including at higher mass resolving power. A common data display is called a deuterium uptake plot. Software to process the voluminous data generated even when looking at only the molecular ions is widely available; below we discuss how the data production ramps rapidly upward.

Figure 2: a The two exchange regimes, which are the rapid side-chain and dynamic region exchanges, and then the more slowly exchanging structured regions. This sample can be analyzed directly or can be digested using the usual methods into smaller peptide lengths that can be separated and analyzed. Figure adapted with permission from reference Figure 2b illustrates the quenching reaction that stops the exchange reaction. Quenching usually occurs when the pH and the temperature are dropped.

This change in conditions may not be perfect, and some back exchange can occur, especially for the very readily exchanged hydrogen—deuterium. The back exchange must also be considered when the sample is further processed for other mass spectral measurements. Figure 2b shows that the sample can be unfolded and then digested using relatively standard techniques. Pepsin or other proteases are used for proteolysis, and the quenched exchange must be maintained through the process. Various forms of liquid chromatography LC are used to separate the individual peptides, which are then analyzed by MS, often with electrospray ionization ESI.

The extent of deuterium exchange can then be established in each of the peptide fragments by comparison of mass expected and mass observed, illustrating the progressively more expansive data processing needs.

Exchange can be assessed, then, in molecular ions or in smaller parts derived from the larger structure. On reflection, it should be obvious that the former represents the total of all exchange reactions occurring, and may not provide information relevant to specific regions of a larger structure.

Probing the exchange reactivity in regions is made possible by the proteolysis illustrated in Figure 2b. Is it possible to determine the amount of deuterium exchange into individual amino acids or small chains of 2—5 amino acids? Certainly being able to assemble such information, residue by residue, over an entire structure would provide a detailed examination of its structure. The mass shift in each ion should be readily discernible, so the problem becomes how to take ionic chains of amino acids and dissociate them.

But it is also known that hydrogen can scramble within the ion as a result of the CID energization, rendering this specific process unsatisfactory. Studies show that electron-capture dissociation and electron-transfer dissociation can, under some conditions, provide the requisite dissociation without scrambling.

The extent of H—D exchange can then be probed residue by residue. For proteins containing hundreds or thousands of residues, the management of the data can become a complex problem. Insights into how to process and interpret this data are continually being developed and refined 12, Reviews over the past 10 years reflect the development of the method, but also different perspectives as it engages the attention of other research communities.

Topics covered in the reviews include mathematical modeling of the kinetics, the display and interpretation of the datasets, new approaches and challenges, including automated workflows and the incorporation of microfluidics, and finally, the breadth and meaning of the conclusions 15— The method is described in general science articles 20 and even a few videos can be found online The information available from H—D exchange MS extends to studies of changes in protein structure as the result of binding to an inhibitor, protein—protein interactions, and folding and unfolding dynamics.

All of this information is revealed in the plots of deuterium uptake with time for the protein as a whole, and for its subregions. Nothing in MS stays simple for long, and H—D exchange MS is a perfect example of a simple concept amplified through insightful experiments that is now being used by a broad range of scientific research and application communities.

As this column was being prepared, the obituary for Virgil Woods appeared in the Journal of the American Society for Mass Spectrometry Woods was involved in the automation of H—D exchange MS dating back to As in much of mass spectrometry today, the capabilities that we sometimes take for granted today can be traced directly to the insight, capabilities, and persistence of earlier researchers.

Urey, F. Brickwedde, and G. Murphy, Phys. Rylander, S. Meyerson, and H. Jump to site search. You do not have JavaScript enabled. Please enable JavaScript to access the full features of the site or access our non-JavaScript page.

Issue 5, Setner , a M. Wierzbicka , a L. Jerzykiewicz , a M. Lisowski a and Z. You have access to this article. Ultrahigh mass resolution enables resolution of dozens of peptides, and is essential for extending the technique to large proteins and complexes. Hydrogen—Deuterium Exchange. Last modified on 7 June